Patent US5498531A successfully refreshed from Google Patents.
US5498531A
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Intron-mediated recombinant techniques and reagents
Assignee: Harvard University
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Filing: 1993-09-10
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Granted: 1996-03-12
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Expires: 2013-09-10
Abstract
Abstract The present invention makes available methods and reagents for novel manipulation of nucleic acids. As described herein, the present invention makes use of the ability of intronic sequences, such as derived from group I, group II, or nuclear pre-mRNA introns, to mediate specific cleavage and ligation of discontinuous nucleic acid molecules. For example, novel genes and gene products can be generated by admixing nucleic acid constructs which comprise exon nucleic acid sequences flanked by intron sequences that can direct trans-splicing of the exon sequences to each other. The flanking intronic sequences can, by intermolecular complementation, form a reactive complex which promotes the transesterification reactions necessary to cause the ligation of discontinuous nucleic acid sequences to one another, and thereby generate a recombinant gene comprising the ligated exons.
Inventors
- 👤 Kevin A. Jarrell
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-05 16:21
CPC Classifications
C; C12; C12N; C12N15/00; C12N15/09; C12N15/63; C12N15/79; C12N15/85; C07; C07K; C07K16/00; C12N15/10; C12N2830/00; C12N2830/42; C12N2840/00; C12N2840/44; C12N2840/445
Claims Analysis
Total Claims: 43
Claim 1
Independent Claim
210 words
A combinatorial method for generating a variegated population of ribonucleic acids encoding chimeric gene products, comprising admixing a variegated set of splicing constructs under trans-splicing reaction conditions, each splicing construct comprising a ribonucleic acid represented by the general formula (3'IVS)-EX-(5'IVS), wherein EX represents an exon which is intended to be present in a chimeric ribonucleic acid, said exon having a 5' exon end and a 3' exon end, (3'IVS) is absent or represents a 3' fragment of an intron, which 3' intron fragment is covalently attached to the 5' exon end of said exon by a phosphodiester bond, and (5'IVS) is absent or represents a 5' fragment of an intron, which 5' intron fragment is covalently attached to the 3' exon end of said exon by a phosphodiester bond, with the proviso that at least a portion of said splicing constructs comprise internal exons having both of said 3' and 5' intron fragments; wherein said exons of said set of splicing constructs comprise a variegated population of ribonucleic acids, and said trans-splicing reaction conditions comprise conditions in which 3' and 5' intron fragments of different splicing constructs reconstitute a functional intron through intermolecular complementation and ligate said exons to generate a variegated population of ribonucleic acids encoding chimeric gene products.
Claim 2
Dependent Claim
210 words
A combinatorial method for generating a variegated population of ribonucleic acids encoding chimeric gene products, comprising admixing a variegated set of splicing constructs under trans-splicing reaction conditions, each splicing construct comprising a ribonucleic acid represented by the general formula (3'IVS)-EX-(5'IVS), wherein EX represents an exon which is intended to be present in a chimeric ribonucleic acid, said exon having a 5' exon end and a 3' exon end, (3'IVS) is absent or represents a 3' fragment of an intron, which 3' intron fragment is covalently attached to the 5' exon end of said exon by a phosphodiester bond, and (5'IVS) is absent or represents a 5' fragment of an intron, which 5' intron fragment is covalently attached to the 3' exon end of said exon by a phosphodiester bond, with the proviso that at least a portion of said splicing constructs comprise internal exons having both of said 3' and 5' intron fragments; wherein said exons of said set of splicing constructs comprise a variegated population of ribonucleic acids, and said trans-splicing reaction conditions comprise conditions in which 3' and 5' intron fragments of different splicing constructs reconstitute a functional intron through intermolecular complementation and ligate said exons to generate a variegated population of ribonucleic acids encoding chimeric gene products.
Claim 3
Dependent Claim
36 words
The method of claim 1, wherein said exons are randomly trans-spliced to each other to generate a library of combinatorial genes comprising, for every N different internal exons, N y different genes having y internal exons.
Claim 4
Dependent Claim
21 words
The method of claim 1, wherein at least a portion of said exons are spliced to each other in predetermined order.
Claim 5
Dependent Claim
66 words
The method of claim 1, wherein said 3' and 5' intron fragments of said splicing constructs comprise group II intron fragments including, i) an exon binding site, and ii) a branch site acceptor comprising an activated nucleophile for forming a phosphodiester bond with a 5' intron end of said 5' intron fragment and for cleaving said 5' intron fragment from the 3' end of said exon.
Claim 6
Dependent Claim
29 words
The method of claim 4, wherein said group II intron fragments further comprise at least a portion of a group II domain V sufficient to reconstitute said functional intron.
Claim 7
Dependent Claim
45 words
The method of claim 4, wherein said trans-splicing reaction conditions further comprise admixing with said splicing constructs at least a portion of a domain V of a group II intron sufficient to interact with said 3' and 5' intron fragments and reconstitute said functional intron.
Claim 8
Dependent Claim
49 words
The method of claim 1, wherein said 3' and 5' intron fragments of said splicing constructs comprise group I intron fragments including an internal guide sequence, a GTP-binding site, and a 3' terminal G located in said 3' intron fragment immediately adjacent said 5' exon end of said exon.
Claim 9
Dependent Claim
55 words
The method of claim 1, wherein said 3' and 5' intron fragments of said splicing constructs comprise nuclear pre-mRNA intron fragments including a 5' splice junction sequence, a 3' splice junction sequence, and a branchpoint sequence; and said trans-splicing reaction conditions include admixing, with said splicing constructs, adenosine triphosphate (ATP) and small nuclear ribonucleoproteins (snRNPs).
Claim 10
Dependent Claim
25 words
The method of claim 8, wherein said snRNPs comprise a U1 snRNP, a U2 snRNP, a U4 snRNP, a U5 snRNP, and a U6 snRNP.
Claim 11
Dependent Claim
22 words
The method of claim 1 further comprising the step of reverse transcribing said variegated population of ribonucleic acids encoding chimeric gene products.
Claim 12
Dependent Claim
202 words
A combinatorial method for generating a variegated population of ribonucleic acids encoding chimeric gene products, comprising admixing a variegated set of splicing constructs under trans-splicing conditions, each splicing construct comprising a ribonucleic acid represented by the general formula (3'IVS)-EX-(5'IVS), wherein EX represents an exon, said exon having a 5' exon end and a 3' exon end, (3'IVS) is absent or represents a 3' fragment of a group II intron, which 3' intron fragment includes a branch acceptor site and at least a portion of a domain V, and is covalently attached to the 5' exon end of said exon by a phosphodiester bond, and (5'IVS) is absent or represents a 5' fragment of a group II intron, which 5' intron fragment includes an exon binding site, and is covalently attached to the 3' exon end of said exon by a phosphodiester bond, with the proviso that at least a portion of said splicing constructs comprise internal exons having both of said 3' and 5' intron fragments; wherein said splicing reaction conditions comprise reaction conditions in which said 3' and 5' intron fragments reconstitute a functional intron through intermolecular complementation and trans-splice said exons to generate a variegated library of chimeric ribonucleic acids.
Claim 13
Dependent Claim
202 words
A combinatorial method for generating a variegated population of ribonucleic acids encoding chimeric gene products, comprising admixing a variegated set of splicing constructs under trans-splicing conditions, each splicing construct comprising a ribonucleic acid represented by the general formula (3'IVS)-EX-(5'IVS), wherein EX represents an exon, said exon having a 5' exon end and a 3' exon end, (3'IVS) is absent or represents a 3' fragment of a group II intron, which 3' intron fragment includes a branch acceptor site and at least a portion of a domain V, and is covalently attached to the 5' exon end of said exon by a phosphodiester bond, and (5'IVS) is absent or represents a 5' fragment of a group II intron, which 5' intron fragment includes an exon binding site, and is covalently attached to the 3' exon end of said exon by a phosphodiester bond, with the proviso that at least a portion of said splicing constructs comprise internal exons having both of said 3' and 5' intron fragments; wherein said splicing reaction conditions comprise reaction conditions in which said 3' and 5' intron fragments reconstitute a functional intron through intermolecular complementation and trans-splice said exons to generate a variegated library of chimeric ribonucleic acids.
Claim 14
Dependent Claim
69 words
The method of claim 11, wherein said branch acceptor site comprises a stem loop structure of a group II domain VI having a branch nucleotide disposed in said 3' intron fragment in the range of 5 to 10 nucleotides from said 5' exon end of said exon and oriented to facilitate nucleophilic attack of its 2'-hydroxyl on a phosphodiester at a 3' exon end of another of said exons.
Claim 15
Dependent Claim
24 words
The method of claim 12, wherein said branch nucleotide is an unbase-paired adenosine residue bulging from said stem loop structure of said domain VI.
Claim 16
Dependent Claim
36 words
The method of claim 11, wherein said exon binding site comprises from 3 to 8 consecutive nucleotides complementary in sequence to an internal binding sequence located within said exon and proximate to said 3' exon end.
Claim 17
Dependent Claim
25 words
The method of claim 11, wherein said 5' intron fragment comprises intron domains V and VI, and said 3' intron fragment comprises intron domains I-III.
Claim 18
Independent Claim
272 words
A method of isolating a gene for chimeric gene product having a desired activity, comprising the steps of: (i) admixing a variegated population of splicing constructs under trans-splicing reaction conditions, each splicing construct comprising a ribonucleic acid represented by the general formula (3'IVS)-EX-(5'IVS), wherein EX represents an exon, said exon having a 5' exon end and a 3' exon end, (3'IVS) is absent or represents a 3' fragment of an intron, which 3' intron fragment is covalently attached to the 5' exon end of said exon by a phosphodiester bond, and (5'IVS) is absent or represents a 5' fragment of an intron, which 5' intron fragment is covalently attached to the 3' exon end of said exon by a phosphodiester bond, with the proviso that at least a portion of said splicing constructs comprise internal exons having both of said 3' and 5' intron fragments; wherein said trans-splicing reaction conditions comprise reaction conditions in which said 3' and 5' intron fragments reconstitute a functional intron through intermolecular complementation, which functional intron, under said trans-splicing conditions, causes ligation of said exons to generate a combinatorial library of chimeric ribonucleic acids; (ii) generating, from said library of chimeric ribonucleic acids, a library of replicable expression vectors; (iii) transforming suitable host cells with said library of replicable expression vectors; (iv) culturing said transformed host cells under conditions causing expression of said library of expression vectors to generate a library of gene products; (v) detecting, in said library of gene products, individual gene products having a desired activity; and (vi) isolating, from said library of expression vectors, genes for the gene products detected in step (v).
Claim 19
Independent Claim
272 words
A method of isolating a gene for chimeric gene product having a desired activity, comprising the steps of: (i) admixing a variegated population of splicing constructs under trans-splicing reaction conditions, each splicing construct comprising a ribonucleic acid represented by the general formula (3'IVS)-EX-(5'IVS), wherein EX represents an exon, said exon having a 5' exon end and a 3' exon end, (3'IVS) is absent or represents a 3' fragment of an intron, which 3' intron fragment is covalently attached to the 5' exon end of said exon by a phosphodiester bond, and (5'IVS) is absent or represents a 5' fragment of an intron, which 5' intron fragment is covalently attached to the 3' exon end of said exon by a phosphodiester bond, with the proviso that at least a portion of said splicing constructs comprise internal exons having both of said 3' and 5' intron fragments; wherein said trans-splicing reaction conditions comprise reaction conditions in which said 3' and 5' intron fragments reconstitute a functional intron through intermolecular complementation, which functional intron, under said trans-splicing conditions, causes ligation of said exons to generate a combinatorial library of chimeric ribonucleic acids; (ii) generating, from said library of chimeric ribonucleic acids, a library of replicable expression vectors; (iii) transforming suitable host cells with said library of replicable expression vectors; (iv) culturing said transformed host cells under conditions causing expression of said library of expression vectors to generate a library of gene products; (v) detecting, in said library of gene products, individual gene products having a desired activity; and (vi) isolating, from said library of expression vectors, genes for the gene products detected in step (v).
Claim 20
Dependent Claim
35 words
The method of claim 16, wherein said 3' and 5' intron fragments comprise group II intron fragments including an exon binding site, a branch acceptor site, and at least a potion of a domain V.
Claim 21
Dependent Claim
41 words
The method of claim 16, wherein said 3' and 5' intron fragments comprise group I intron fragments including an internal guide sequence, a GTP-binding site, and a guanosine nucleotide located in said 3' intron fragment immediately adjacent said 5' exon end.
Claim 22
Dependent Claim
53 words
The method of claim 16, wherein (i) said 3' and 5' intron fragments comprise nuclear pre-mRNA intron fragments including a 5' splice junction sequence, a 3' splice junction sequence, and a branchpoint sequence; and (ii) said trans-splicing reaction conditions include admixing, with said splicing constructs, adenosine triphosphate (ATP) and small nuclear ribonucleoproteins (snRNPs).
Claim 23
Dependent Claim
12 words
The method of claim 16, wherein said gene product is a polypeptide.
Claim 24
Dependent Claim
12 words
The method of claim 16, wherein said gene product is a ribozyme.
Claim 25
Dependent Claim
36 words
The method of claim 16, wherein said desired activity is an enzymatic activity and detecting said desired activity comprises detecting an enzymatic alteration of a substrate by individual gene products of said library of gene products.
Claim 26
Dependent Claim
39 words
The method of claim 16, wherein said desired activity is a binding affinity for a target molecule and detecting said desired activity comprises detecting binding to said target molecule by individual gene products of said library of gene products.
Claim 27
Dependent Claim
17 words
The method of claim 23, wherein said gene product is an antibody or functional binding fragment thereof.
Claim 28
Dependent Claim
17 words
The method of claim 16, wherein said library of replicable expression vectors comprises a phage display library.
Claim 29
Dependent Claim
137 words
A splicing construct comprising a nucleic acid represented by the general Formula (3'IVS)-EX-(5'IVS), wherein EX represents an exon, said exon having a 5' exon end and a 3' exon end, (3'IVS) represents a 3' fragment of an intron not normally associated with said exon, which 3' intron fragment is covalently attached to the 5' exon end of said exon by a phosphodiester bond, and (5'IVS) represents a 5' fragment of an intron not normally associated with said exon, which 5' intron fragment is covalently attached to the 3' exon end of said exon by a phosphodiester bond, wherein said exon is discontinuous with any nucleic acid sequences other than said 3' and 5' intron fragments, and said 3' and 5' intron fragments can, through intermolecular complementation, mediate trans-splicing reactions between two or more of said splicing constructs.
Claim 30
Dependent Claim
137 words
A splicing construct comprising a nucleic acid represented by the general Formula (3'IVS)-EX-(5'IVS), wherein EX represents an exon, said exon having a 5' exon end and a 3' exon end, (3'IVS) represents a 3' fragment of an intron not normally associated with said exon, which 3' intron fragment is covalently attached to the 5' exon end of said exon by a phosphodiester bond, and (5'IVS) represents a 5' fragment of an intron not normally associated with said exon, which 5' intron fragment is covalently attached to the 3' exon end of said exon by a phosphodiester bond, wherein said exon is discontinuous with any nucleic acid sequences other than said 3' and 5' intron fragments, and said 3' and 5' intron fragments can, through intermolecular complementation, mediate trans-splicing reactions between two or more of said splicing constructs.
Claim 31
Dependent Claim
23 words
The nucleic acid construct of claim 26, wherein said exon comprises an open reading frame encoding at least a portion of a protein.
Claim 32
Dependent Claim
20 words
The nucleic acid construct of claim 27, wherein said protein is naturally encoded by genomic DNA of a eukaryotic cell.
Claim 33
Dependent Claim
21 words
The nucleic acid construct of claim 26, wherein said exon comprises at least a portion of a ribozyme nucleic acid sequence.
Claim 34
Dependent Claim
19 words
The nucleic acid construct of claim 26, wherein said 3' and 5' intron fragments comprise group II intron fragments.
Claim 35
Dependent Claim
19 words
The nucleic acid construct of claim 26, wherein said 3' and 5' intron fragments comprise group I intron fragments.
Claim 36
Dependent Claim
19 words
The nucleic acid construct of claim 26, wherein said 3' and 5' intron fragments comprise nuclear pre-mRNA intron fragments.
Claim 37
Dependent Claim
141 words
A library of splicing constructs comprising a variegated population of nucleic acids each represented by the general formula (3'IVS)-EX-(5'IVS), wherein EX represents an exon, said exon having a 5' exon end and a 3' exon end, (3'IVS) represents a 3' fragment of an intron not normally associated with said exon, which 3' intron fragment is covalently attached to the 5' exon end of said exon by a phosphodiester bond, and (5'IVS) represents a 5' fragment of an intron not normally associated with said exon, which 5' intron fragment is covalently attached to the 3' exon end of said exon by a phosphodiester bond, wherein said 3' and 5' intron fragments can, through intermolecular complementation, cause trans-splicing of two or more exons to each other, and said variegated population of nucleic acids is heterogeneous with respect to nucleotide sequences of said exon.
Claim 38
Dependent Claim
141 words
A library of splicing constructs comprising a variegated population of nucleic acids each represented by the general formula (3'IVS)-EX-(5'IVS), wherein EX represents an exon, said exon having a 5' exon end and a 3' exon end, (3'IVS) represents a 3' fragment of an intron not normally associated with said exon, which 3' intron fragment is covalently attached to the 5' exon end of said exon by a phosphodiester bond, and (5'IVS) represents a 5' fragment of an intron not normally associated with said exon, which 5' intron fragment is covalently attached to the 3' exon end of said exon by a phosphodiester bond, wherein said 3' and 5' intron fragments can, through intermolecular complementation, cause trans-splicing of two or more exons to each other, and said variegated population of nucleic acids is heterogeneous with respect to nucleotide sequences of said exon.
Claim 39
Dependent Claim
25 words
The library of splicing constructs of claim 33, wherein exons of said variegated population of nucleic acids encode portions of at least one thrombolytic protein.
Claim 40
Dependent Claim
24 words
The library of splicing constructs of claim 33, wherein exons of said variegated population of nucleic acids encode portions of at least one antibody.
Claim 41
Dependent Claim
177 words
A kit for humanizing an antibody by combinatorial intron-mediated ligation, comprising a library of human framework region (FR) constructs each represented by the general formula (3'IVS)-FR-(5'IVS), wherein FR represents an exon encoding a human framework region selected from the group consisting of FR1, FR2, FR3 and FR4, said exon having a 5' exon end and a 3' exon end, (3'IVS) is absent or represents a 3' fragment of an intron, which 3' intron fragment is covalently attached to the 5' exon end of said exon by a phosphodiester bond, and (5'IVS) is absent or represents a 5' fragment of an intron, which 5' intron fragment is covalently attached to the 3' exon end of said exon by a phosphodiester bond, with the proviso that one or both of said 3' and 5' intron fragments are present in each FR construct; wherein said 3' and 5' intron fragments can, through intermolecular complementation, mediate trans-splicing between said exon and a complementarity determining region (CDR) of a construct comprising CDR-encoding nucleic acid having 3' and 5' intron fragments attached thereto.
Claim 42
Dependent Claim
177 words
A kit for humanizing an antibody by combinatorial intron-mediated ligation, comprising a library of human framework region (FR) constructs each represented by the general formula (3'IVS)-FR-(5'IVS), wherein FR represents an exon encoding a human framework region selected from the group consisting of FR1, FR2, FR3 and FR4, said exon having a 5' exon end and a 3' exon end, (3'IVS) is absent or represents a 3' fragment of an intron, which 3' intron fragment is covalently attached to the 5' exon end of said exon by a phosphodiester bond, and (5'IVS) is absent or represents a 5' fragment of an intron, which 5' intron fragment is covalently attached to the 3' exon end of said exon by a phosphodiester bond, with the proviso that one or both of said 3' and 5' intron fragments are present in each FR construct; wherein said 3' and 5' intron fragments can, through intermolecular complementation, mediate trans-splicing between said exon and a complementarity determining region (CDR) of a construct comprising CDR-encoding nucleic acid having 3' and 5' intron fragments attached thereto.
Claim 43
Dependent Claim
33 words
The kit of claim 36, wherein said FR constructs are selected for sequential ligation of said exons with said CDR-encoding nucleic acids to form a ribonucleic acid represented by a general formula 5'-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-3'.
References Cited (Backward Citations)
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
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| 3.5 Years (Year 4) | 1999-03-12 | 1999-09-11 | 2000-03-12 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 2003-03-12 | 2003-09-11 | 2004-03-12 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2007-03-12 | 2007-09-11 | 2008-03-12 | $7700.00 | Window Closed / Lapsed |